The identification of genetic pathways controlling cardiac chamber formation is likely to illuminate the molecular mechanisms responsible for many cardiac structural birth defects. The cardiac homeodomain transcription factor gene Nkx2-5 is known to play a key role in cardiac development: Nkx2-5 is essential for normal chamber formation in mice, and mutations in human Nkx2-5 underlie familial cases of congenital heart disease. Despite the clear importance of Nkx2-5 in the cardiac regulatory hierarchy, the downstream components of the Nkx2-5 pathway and their contributions to cardiac chamber formation are not well understood. The PIs hypothesize that the essential effector genes for cardiac chamber formation are evolutionarily conserved components of the Nkx2-5 pathway. To test this hypothesis, they propose a two-year exploratory project that combines the expertise of the Yelon and Harvey laboratories for a comparative genetic analysis of Nkx2-5 function in zebrafish and mouse. Specifically, they will begin by characterizing the molecular anatomy of the zebrafish heart as a foundation for comparative analysis. Applying this knowledge, they will compare the loss-of-function phenotypes for the Nkx2-5 gene family in zebrafish and mouse. Then, taking advantage of advances in both mouse and zebrafish genomics, they will combine microarray analysis and comparative gene expression studies to identify genes downstream of Nkx2-5 in both species. Finally, with attention focused on conserved pathway components, they will utilize zebrafish reverse genetics to test their roles during cardiac chamber formation. Together, the proposed experiments will reveal conserved roles and components of the Nkx2-5 pathway. Furthermore, by capitalizing on the complementary advantages of two model organisms, the PIs will develop a new approach for functional annotation of cardiac gene expression profiles.